End-stage renal disease (ESRD) upsets the lives of over half a million people in the United States, with an incidence of over 100,000 new cases per year and a mortality rate of approximately 88,000 people each year. The Veteran's Affairs cares for approximately 30,000 veterans with ESRD. The costs to treat ESRD, approximately $39 billion in 2008, continue to grow. The most common option chosen for renal replacement therapy is hemodialysis; access for hemodialysis is performed by either arteriovenous fistula (AVF), arteriovenous graft (AVG), or temporary catheter placement. Although fistulae take time to mature, e.g. dilate, thicken and increase flow, prior to beginning dialysis, AVF continue to be the preferred mode of access, with superior long term results compared to AVG and catheter access. Despite the superiority of AVF access compared to its alternatives, AVF are still far from perfect. AVF fail to mature in approximately 20-50% of cases, with many of these AVF requiring some therapeutic intervention to mature successfully. In fact, these poor clinical results demonstrate that AVF defy all the rules of vascular surgery; despite their desirable characteristics of short length, large diameter, high flow, and low resistance runoff, AVF exhibit the worst patency of any procedure performed by vascular surgeons. The poor patency of AVF clearly reflects our imperfect understanding of the biology of venous remodeling leading to AVF maturation and the unmet medical need for novel approaches to enhance AVF maturation and subsequent fistula usage. Several members of the Ephrin-Eph pathway have recently been described as developmentally specified critical determinants of vessel identity, with Ephrin-B2 ligand an embryonic determinant of arteries and Eph-B4 receptor an embryonic determinant of veins. However, it is currently not established whether Eph- B4 is functional in adult veins, and whether the Ephrin-Eph pathway plays a mechanistic role during AVF adaptation to the arterial environment. We hypothesize that increased Eph-B4 expression and/or activation (as assessed by phosphorylation) mediates increased AVF wall diameter and thickening during normal AVF maturation. This is a different mechanism than by which Eph-B4 mediates vein graft adaptation. We will test our hypothesis with these aims: Aim I: To determine whether Eph-B4 mediates diameter expansion and/or wall thickening during normal AVF maturation. Our hypothesis, based on our preliminary data, is that increased expression and/or activation of the venous specification gene Eph-B4 leads to normal AVF diameter expansion and wall thickening and fistula maturation. We will examine surgical specimens derived from patent human AVF undergoing surgical revision or ligation to determine how AVF diameter and/or wall thickness correlate with Eph-B4 expression and activation. We will test this hypothesis by directly increasing and decreasing Eph-B4 signaling in our mouse AVF model and correlating vessel identity with AVF diameter and wall thickness. Aim II: To determine how shear stress regulates Eph-B4 phosphorylation and expression. Our hypothesis is that shear stress promotes AVF maturation by stimulating Eph-B4 signaling and expression in endothelial cells; in this way, changes in shear stress lead to AVF dilation and wall thickening. We will test this hypothesis by altering shear stress in our in vitro flow models, using both whole vein and endothelial cell flow models, and show that arterial magnitudes of shear stress stimulate Eph-B4 phosphorylation and expression. Aim III: To determine how Eph-B4 signaling regulates vascular cell function. Our hypothesis is that Eph- B4 phosphorylation on tyrosine-774 is a critical regulator of Eph-B4 downstream function in vascular cells. We will test this hypothesis by examining the differential effects of wild type and mutated Eph-B4 on endothelial and smooth muscle cell signal transduction as well as cell proliferation and migration.